All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.
Immune therapy involves the exposure of components of the immune system to various elements (cytokines, disease associated antigens and natural metabolites) to combat disease processes in which a dysregulated immune response is thought to play a role. Immune dysregulation is thought to play a major part in the pathogenesis or disease course of a great number of disease processes, including various neoplastic, inflammatory, infectious and genetic entities.
These disorders can be perceived as a dysbalance between pro-inflammatory (Th1) and anti-inflammatory (Th2) cytokines, and few of them are described herein below.
Natural killer T (NKT) cells are a highly conserved subset of lymphocytes that can be broadly categorized into two groups [Brigl, M. and Brenner M. B. Annu. Rev. Immunol. 22: 817-90 (2004)]. In mice, invariant NKT (iNKT; also referred to as classical or type I NKT) cells express a conserved invariant αβ T cell receptor (TCR), encoded by the Vα14 (Vα24 in humans) and Jα18 gene segments, paired with a set of Vβ chains [Bendelac. A. et al. Annu. Rev. Immunol. 25:297-336 (2007)]. These cells are CD1d-dependent, reactive to α-Galctosyl-ceramide (α-GalCer), and are potent producers of both interleukin 4 (IL-4) and interferon gamma (IFN-γ). Non-classical or type II NKT cells, which use variable TCRs, are distinct from type I NKT cells and have been identified in both humans and mice [Chiu, Y. H. et al. Nat. Immunol. 3:55-60 (2002); Godfrey, D. I. et al. Science, 306: 1687-9 (2004)]. Type II (non-classical) NKT cells, which may be either CD161-positive or negative, are also CD1d-dependent and potent IL-4 and IFN-γ producers. However they have more diversely structured TCR α and β chains and are not α-Gal-Cer reactive [Brigl, M. and Brenner M. B. Annu. Rev. Immunol. 22: 817-90 (2004); Bendelac. A. et al. Annu. Rev. Immunol. 25:297-336 (2007)].
The CD1 glycoprotein family resembles classical peptide antigen presenting molecules except that the nonpolar, hydrophobic, antigen-binding groove has evolved to present lipid antigens [Brigl, M. and Brenner M. B. Annu. Rev. Immunol. 22: 817-90 (2004)]. Antigens presented by CD1d and/or the level at which they are presented can have profound effects on immune regulation of autoimmune, infectious, and malignant disorders [Brutkiewicz, R. R. J. Immunol. 177: 769-75 (2006); Brutkiewicz, R. R. et al. Crit. Rev. Immunol. 23: 403-19 (2003); Van Kaer, L. Curr. Opin. Immunol. 19: 354-64 (2007)]. Abnormalities in the number and function of NKT cells have been observed in patients with autoimmune diseases and in a variety of mouse strains genetically predisposed for the development of autoimmune diseases [Miyake, S and Yamamura, T. Curr. Drug Targets Immune Endocr. Metabol. Disord. 5: 315-22 (2005)]. Targeting CD1d-mediated antigen presentation can serve as a novel approach for intervening in autoimmune diseases [Brutkiewicz, R. R. et al. Crit. Rev. Immunol. 23: 403-19 (2003); Singh, A. K. et al. J. Exp. Med. 194: 1801-11 (2001); Sandberg, J. K. and Ljunggren, H. G. Trends Immunol. 26: 347-9 (2005)].
Type I and type II NKT cells share some important characteristics. However, their distinct developmental and selection requirements indicate that they have distinct specificities [Godfrey, D. I. et al. Science, 306: 1687-9 (2004)]. As both type I and type II NKT cells are CD1d-dependent, any phenotype observed in CD1d-deficient mice might be due to a deficiency in type II NKT cells rather than type I NKT cells [Huber, S. et al. J. Immunol. 170: 3147-53 (2003); Exley, M. A. and Koziel, M. J. Hepatology, 40:1033-40 (2004); Dao, T. et al. Eur. J. Immunol. 34: 3542-52 (2004)], and the functional relationship between these two cell types is unclear. To this end, the combined use of TCR Jα18-deficient mice and CD1d-deficient mice is useful to distinguish between type I and type II NKT cells at the functional level in vivo. Although iNKT (invariant NKT) cells have been shown to be involved in autoimmune diseases, infectious diseases, and asthma as well as in the antitumor immune responses, not much is known about type II NKT cells.
NKT lymphocytes are influenced by a variety of self ligands and environmental stimuli, including disease target antigens, antigen presenting cells, co-stimulatory signals, soluble factors, and effector cells [Godfrey, D. I. and Kronenberg, M., J. Clin. Invest. 114: 1379-88 (2004); Smyth, M. J. et al. Curr. Opin. Immunol. 14:165-71 (2002)]. NKT cell functions are controlled by affinity thresholds for glycosphingolipid antigens that play an important role in cell activation16. Activation and development of NKTs are guided by information provided by glycosphingolipid metabolic pathways [Hammond, K. J. and Godfrey, D. I. Tissue Antigens, 59: 353-63 (2002); Kronenberg, M. Annu. Rev. Immunol. 23: 877-900 (2005)].
Currently, the only efficient method to selectively stimulate NKT cells in vivo is the sea sponge-derived agent αGalCer [Van Kaer, L. Nat. Rev. Immunol. 5: 31-42 (2005)]. Tetrameric forms of CD1d molecules bound to αGalCer, which have sufficient affinity for the TCR of iNKT cells, allow for the detection of these cells by flow cytometry [Taniguchi, M. et al. Annu. Rev. Immunol. 21: 483-513 (2003); Bezbradica, J. S. et al. J. Immunol. 174: 4696-705 (2005)]. Administration of αGalCer results in potent activation of NKT cells, rapid and robust cytokine production, and further activation of a variety of cells of the innate and adaptive immune systems [Van Kaer, L. Nat. Rev. Immunol. 5: 31-42 (2005)]. Transient administration of αGalCer induces both IL-4 and IFN-γ secretion, while repeated administration favors production of Th2 cytokines [Miyake, S and Yamamura, T. Curr. Drug Targets Immune Endocr. Metabol. Disord. 5: 315-22 (2005); Yamamura, T. et al. Curr. Top Med. Chem. 4: 561-7 (2004)]. OCH, a sphingosine-truncated analog of αGalCer, was shown to be a potential therapeutic reagent for a variety of Th1-mediated autoimmune diseases through its selective induction of Th2 cytokines from iNKT cells [Oki, S. et al. J. Clin. Invest. 113: 1631-40 (2004)].
Both rodent and human iNKT cells have been reported to recognize αGalCer in the context of CD1d. Therefore, activated iNKT cells play a pivotal role in modulating many aspects of the innate immune response within the liver during a diverse array of pathological processes [Ajuebor, M. N. and Swain, M. G. Immunology, 105: 137-43 (2002)]. However, αGalCer has been shown to be hepatotoxic in mice, which has limited its use in human testing [Osman, Y. et al. Eur. J. Immunol. 30: 1919-28 (2000); Biburger, M. and Tiegs, G. J. Immunol. 175: 1540-50 (2005)].
While the responses to antigen presentation can be complex, the process of antigen binding, presentation, and recognition by T cell receptors is fundamentally a series of molecular recognition events. In recent years, glycolipid presentation by CD1 proteins has emerged as an important aspect of antigen recognition [Brigl, M. and Brenner M. B. Annu. Rev. Immunol. 22: 817-90 (2004)].
Several glycosphingolipids and phospholipids derived from mammalian, bacterial, protozoan, and plant species have been identified as possible natural ligands for NKT cells [Zajonc, D. M. et al. Nat. Immunol. 6: 810-8 (2005); Tsuji, M. Cell Mol. Life Sci. 63: 1889-98 (2006)]. A semi-invariant αβTCR can recognize iGb3, a mammalian glycosphingolipid, as well as a microbial alpha-glycuronylceramide found in the cell wall of Gram-negative, lipopolysaccharide-negative bacteria [Zhou, D. Curr. Protein Pept. Sci. 7: 325-33 (2006)]. iGb3 has been suggested as a candidate recognized by NKT cells under pathophysiological conditions, such as cancer and autoimmune disease [Zhou, D. Curr. Protein Pept. Sci. 7: 325-33 (2006); Hansen, D. S. and Schofield, L. Int. J. Parasitol. 34: 15-25 (2004)]. However, the normal development and function of invariant NKT cells in mice with iGb3 deficiency has been described [Porubsky, S. et al. Proc. Natl. Acad. Sci. U.S.A. 104: 5977-82 (2007); Skold, M. and Behar, S. M. Infect. Immun. 71: 5447-55 (2003); Tupin, E. et al. Nat. Rev. Microbiol. 5: 405-17 (2007); Stanic, A. K. et al. Immunology, 109: 171-84 (2003)].
Alpha-anomeric D-glycosylceramides are unknown in mammals. In contrast to current thinking, β-anomeric GalCer can induce CD1d-dependent biological activities in mice, albeit at lower potency than α-anomeric GalCer. β-anomeric glycolipids may provide a source of weakly reactive self antigens. Binding studies using CD1d tetramers loaded with α-GalCer (C12) demonstrated significant but lower intensity binding to NKT cells when compared with α-GalCer. Glucosylceramide (β-GC), a naturally occurring glycosphingolipid, is a metabolic intermediate in the anabolic and catabolic pathways of glycosphingolipids [Yang, Y. G. et al. Cell Mol. Immunol. 2: 323-9 (2005)]. Its synthesis from ceramide is catalyzed by glucosylceramide synthase [Lalazar, G. et al. Mini Rev. Med. Chem. 6: 1249-53 (2006)]. Inherited deficiency of glucosylceramidase (a lysosomal hydrolase) results in high serum levels of β-GC and Gaucher's disease. These patients have altered humoral and cellular immune profiles, and distorted NKT lymphocyte numbers and functions [Lalazar, G. et al. Mini Rev. Med. Chem. 6: 1249-53 (2006)]. In vitro, CD1d-bound β-GC inhibits NKT cell activation by αGalCer [Ortaldo, J. R. et al. J. Immunol. 172: 943-53 (2004)]. Administration of β-D-GlcCer in vivo attenuated the NKT-mediated damage in concanavalin A (Con A) hepatitis, immune-mediated colitis, and animal models of diabetes [Yang, Y. G. et al. Cell Mol. Immunol. 2: 323-9 (2005); Margalit, M. et. al. Am. J. Physiol. Gastrointest. Liver Physiol. 289: G917-25 (2005); Zigmond, E. et al. Gut 56: 82-9 (2007); Margalit, M. et al. J. Pharmacol. Exp. Ther. 319: 105-10 (2006)].
iNKT cells are influenced by a variety of self ligands and environmental stimuli, including disease target antigens, antigen presenting cells, co-stimulatory signals, soluble factors, and effector cells [Godfrey, D. I. and Kronenberg, M., J. Clin. Invest. 114: 1379-88 (2004); Smith, P. A. et al. AIHA J. (Fairfax, Va.) 63: 284-92 (2002)]. iNKT cell functions are controlled by affinity thresholds for glycosphingolipid antigens that play an important role in cell activation [Stanic, A. K. et al. J. Immunol. 171: 4539-51 (2003)]. However, cellular interactions among NKT cell subsets and their potential role in the regulation of an iNKT cell mediated disease have not been previously investigated.
Abnormalities in the number and function of NKT cells have been observed in patients with autoimmune diseases and in a variety of mouse strains genetically predisposed to the development of autoimmune diseases [Miyake, S. and Yamamura, Curr. Top Microbiol. Immunol. 314: 251-67 (2007)]. Targeting CD1d-mediated antigen presentation can thus serve as a novel approach for intervening in autoimmune diseases [Brutkiewicz, R. R. J. Immunol. 177: 769-75 (2006); Sandberg, J. K. and Ljunggren, H. G. Trends Immunol. 26: 347-9 (2005)].
The Con A model is a widely utilized mouse model that mimics many aspects of human autoimmune hepatitis. It induces massive liver necrosis in mice simultaneously with lymphocyte infiltration in the liver, high levels of apoptotic hepatocytes, and elevated serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The liver injury was confirmed to be NKT cell-mediated [Tiegs, G. Z. Gastroenterol. 45: 63-70 (2007)]. iNKT cells have recently been shown to play a key role in the development of Con A-induced hepatitis: both Jα18−/− and CD1d−/− mice that lack iNKT cells are relatively resistant to Con A-induced hepatic injury [Kaneko, Y. et al. J. Exp. Med. 191: 105-14 (2000); Takeda, K. and Okumura, K. Hum. Cell, 14: 159-63 (2001)]. Signaling through the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway is an important signal pathway that is activated immediately after cytokine stimulation [Darnell, J. E. Jr. et al. Science, 264: 1415-21 (1994)]. Con A-induced hepatitis is largely dependent on interferon IFN-γ/STAT1, as IFN-γ and STAT1-deficient mice are resistant to Con A-induced hepatitis. On the other hand, IL-6 and cytokines that activate STAT3 protect mice against liver damage.
There is therefore a need to provide safe and effective glycolipid analogs for use as immunomodulators. Such novel analogs may preferably modulate activity of iNKT cells and different signal transductions pathways and thereby alleviate immune-related disorders.
The present invention thus describes the synthesis non-natural different analogs of β-GC. One of these analogs, ALIB-97 is characterized by a bulky acyl-residue and by a thiourea bond. Because Con A-induced liver damage is mediated by iNKT cells, the inventors used this model of liver disease to examine the newly synthesized β-glycosphingolipid analogs of the invention. The invention demonstrates that orally administered ALIB-97 is a potent ligand that is effective in alleviating NKT cell-mediated liver damage in Con A-induced hepatitis. This effect was mediated by altered STAT protein expression and suppression of the production of pro-inflammatory cytokines. The effect noted for ALIB-97 was superior to that of the natural glycosphingolipids and comparable to that of dexamethasone.
The inventors have further investigated the potential effect of the novel analogs of the invention on other immune-related disorders using the ob/ob mice that serve as a model for metabolic syndrome and NASH.
Non-alcoholic fatty liver disease (NAFLD) has emerged as a substantial public health concern as it may progresses to non-alcoholic steatohepatitis (NASH) and end stage liver disease [Matteoni, C. A. et al. Gastroenterology, 116: 1413-9 (1999)]. It is commonly associated with clinical features of the metabolic syndrome including type 2 diabetes, obesity and dyslipidemia. Insulin resistance plays an important role in the pathogenesis of NASH. The two-hit model, suggesting an initial lipid accumulation in the hepatocytes, followed by a second hit, was suggested for the development of liver injury.
c-Jun N-terminal kinase (JNK) is a member of the subfamily of mitogen-activated protein kinases (MAPKs), and regulates several cellular responses including differentiation, proliferation, migration, immune reaction, and cell death in response to a diverse range of stimuli [Kodama, Y. and Brenner, D. A. Hepatology, 49: 6-8 (2009)]. Three different genes for JNKs, JNK1, JNK2 and JNK3, are known. JNK3 expression is largely restricted to brain, heart and testis whereas JNK1 and 2 are expressed ubiquitously [Davis, 2000]. Recent studies have suggested that activation of JNK plays a central role in the development of obesity, insulin resistance and steatohepatitis [Hirosumi, 2002; Tuncman, 2006; Schattenberg, 2006]. Recent studies have demonstrated that JNK1 and JNK2 differ in function [Singh, R. et al. Hepatology, 49: 87-96 (2009)] [Schattenberg, 2006]. Both isoforms were found to mediate insulin resistance in high-fat diet (HFD)-fed mice, but distinct effects were found for steatohepatitis. Activation of JNK1 was shown to promote the development of steatohepatitis in the murine methionine- and choline-deficient (MCD) diet [Schattenberg, 2006]. Knockdown of JNK2 improved insulin sensitivity but had no effect on hepatic steatosis and markedly increased liver injury [Singh, R. et al. Hepatology, 49: 87-96 (2009)]. The differential effect of JNK2 knockdown, which is effective on insulin resistance but not on hepatic steatosis, highlights that hepatic steatosis, is more than a mere consequence of insulin resistance. JNK1 might have a direct effect on hepatic lipogenesis that is independent of insulin resistance. The distinct roles for JNK1 and JNK2 in hepatocyte death are still controversial. JNK1 phosphorylates and activates Itch, which induces ubiquitination/degradation of c-FLIP (cellular FLICE-inhibitory protein) and subsequent caspase-8-dependent apoptosis, whereas JNK2 may activate caspase-8 more directly [Chang, 2006; Wang, 2006]. Activation of JNK1/2 seems to be involved in TNF-α-induced insulin resistance, causing phosphorylation of IRS1 at the Ser312 residue. JNK2 (−/−) mice fed a HFD are obese and insulin-resistant, similar to wild-type mice, and have increased liver injury [Singh, R. et al. Hepatology, 49: 87-96 (2009)]. This suggests that JNK1 promotes steatosis and hepatitis, whereas JNK2 inhibits hepatocytes' death by blocking the mitochondrial death pathway.
One of the major pathways that are activated by insulin is the Akt (or PKB) pathway which mediates the effects of insulin on glucose transport, glycogen synthesis, protein synthesis, lipogenesis and suppression of hepatic gluconeogenesis. Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 [Lawlor, 2001]. Akt-2 (PKBβ) is highly expressed in insulin-responsive tissues such as adipose tissue [Walker, 1998]. Exciting new data establishes Akt-2 as an essential gene for the maintenance of normal glucose homeostasis [Cho, 2001]. Mice deficient in Akt-2 display many of the typical features of Type II diabetes mellitus in humans, namely hyperglycemia, elevated blood insulin levels, and insulin resistance in the liver and to a minor extent muscle.
Insulin resistance is characterized by a complex interaction of genetic determinants, lifestyle, ageing and nutritional factors. Visceral obesity is regarded as the major cause of insulin resistance. Visceral obesity has also been defined as an important component of the metabolic syndrome, and increased visceral fat mass contributes to the development of obesity-related disorders such as insulin resistance, non-alcoholic fatty liver disease, hypertension, diabetes and CVD [de Luca, 2008]. Chronic systemic inflammation has been proposed to have an important role in the pathogenesis of obesity-related insulin resistance [Bastard, 2006]. There is strong evidence that adipose tissue not only releases FFA that contributes to insulin resistance in liver and muscle, but also produces a wide range of inflammatory molecules including TNF-α and IL-6 which may have local effects on adipose physiology and also systemic effects on other tissues. Some factors such as leptin, TNF-α, IL-6 and resistin are overproduced during obesity, and conversely the plasma levels of adiponectin are reduced during obesity. Both adipose tissue and the liver have an architectural organization in which metabolic cells (adipocytes or hepatocytes) are in close proximity to immune cells (Kupffer cells or macrophages), forming a suitable environment for continuous and dynamic interactions between immune and metabolic responses in both tissues [Hotamisligil, G. S. Nature, 444: 860-7 (2006)]. TNF-α is overexpressed in the adipose tissue of obese mice supporting a link between obesity, diabetes and chronic inflammation [Hotamisligil, G. S. et al. Science, 259: 87-91 (1993)]. White adipose tissue (WAT) is the site of energy storage and is composed of many cell types, adipocytes being the most abundant. Other cell types present in WAT are included in the stromal-vascular fraction, of which approximately 10% are CD14+CD31+ macrophages [Curat, C. A. et al. Diabetes, 53: 1285-92 (2004)]. The number of macrophages present in WAT is directly correlated with adiposity and with adipocyte size in both human subjects and mice [Curat, C. A. et al. Diabetes, 53: 1285-92 (2004); Weisberg, S. P. et al. J. Clin. Invest. 112: 1796-808 (2003); Xu, H. et al. J. Clin. Invest. 112: 1821-30 (2003)]. Adipocytes secrete a variety of protein signals as well as cytokines and chemokines, such as TNF-α, IL-6 and IL-10. About one third of the IL-6 level in the peripheral circulation originates from adipose tissue [Fernandez-Real, J. M. and Ricart, W. Endocr. Rev. 24: 278-301 (2003)]. With increasing obesity, the contribution of IL-6 from the adipose tissue increases [Trayhurn, P. et al. C. J. Nutr. 136: 1935S-1939S (2006); Kershaw, E. E. and Flier, J. S. J. Clin. Endocrinol. Metab. 89: 2548-56 (2004)]. Additionally, anatomically, visceral abdominal fat is venouslt drained in a portal system to the liver, thus further consolidating the effect of WAT inflammatory processes on hepatic function.
Regulatory T cells (Tregs) are fundamental in controlling various immune responses. The CD4+ regulatory T cells have been categorized into two major subgroups based on their ontogeny: The naturally occurring forkhead box P3 (FOXP3)+CD4+CD25+ regulatory T cells referred as Treg cells and the inducible regulatory T cells, which are generated in the periphery under various tolerogenic conditions [Shevach, E. M. Immunity, 25: 195-201 (2006)].
Both α and β glycosphingolipids have an immune modulatory role and can alter the induction and distribution of Tregs, affecting immune-system dependent disorders [Araki, M. et al. Curr. Med. Chem. 15: 2337-45 (2008); Lalazar, G. et al. Mini Rev. Med. Chem. 6: 1249-53 (2006); Van Kaer, L. Nat. Rev. Immunol. 5: 31-42 (2005)]. This effect is dependent on dendritic cells (DCs) and/or alteration of NKT cells palstiticity [Ilan, Y. et al. Transplantation, 83: 458-67 (2007); Margalit, M. et al. Am. J. Physiol. Gastrointest. Liver Physiol. 289: G917-25 (2005); Zigmond, E. et al. Gut, 56: 82-9 (2007); Onoe, K. et al. Immunol. Res. 38: 319-32 (2007)]. Beta-glycolipids were shown to alleviate the metabolic syndrome in animal models and humans [Margalit, M. et al. J. Pharmacol. Exp. Ther. 319: 105-10 (2006); Zigmond, E. et al. Am. J. Physiol. Endocrinol. Metab. 296: E72-8 (2009); Zigmond, E. et al. Hepatology, 44: 180A (2006)].
The present invention demonstrates the beneficial effect of the novel analogs using this model and thereby the applicability of these novel compounds as immuno-modulating agents. More specifically, the results presented by the invention shed light on a cross talk between the immune system, especially in the adipose tissue and JNK signaling pathway. The data suggest new potential therapeutic targets for insulin resistance and NASH. Adipose tissue-specific CD4+ T regulatory cells are suggested as central players in the interplay between the immune system and the pancreas. They may exert their effect by secreting cytokines or by enhancing chemokines, including adipokine secretion from cells in the relevant microenvironment. Ligands such as the novel analogs of the invention and specifically, ALIB-97, via their oral effect on the induction of CD4+ T regulatory cells may serve as novel approach for the alleviation of insulin resistance and for the treatment of type 2 diabetes and NASH.
Thus, the present invention now provides novel synthetic derivatives of β-glycolipids, and particularly of the compounds of Formulas I, II, III and IV, and demonstrates uses thereof in the treatment of pathologic disorders.
It is therefore an object of the invention to provide novel synthetic derivatives of β-glycolipids, as well as compositions thereof and methods for treating pathologic disorders such as immune-related disorders and neurodegenerative disorders.
These and other objects of the invention will become clearer as the description proceeds.